Abstract Zika virus (ZIKV) is the latest emerging infectious virus raising worldwide attention. ZIKV infection has led to severe diseases, including neurological and congenital Zika syndromes, as represented by microcephaly with fetal damage, fetal and pup death, brain abnormalities, and other malformations. ZIKV may infect pregnant fetuses and persistently exist in neuronal and reproductive tissues for months, resulting in severe tissue damage. Currently, there are no therapeutic agents available to prevent and treat ZIKV-caused severe diseases in humans, calling for the development of novel anti-ZIKV therapeutics for human use. ZIKV envelope (E) protein is a key therapeutic and vaccine target. ZIKV E-specific neutralizing monoclonal antibodies (mAbs) are being developed preclinically and/or tested clinically, but some of them have failed to fully stop vertical transmission of ZIKV. Moreover, the conventional mAbs usually have relatively large size, complicated structure, unstable behavior, and/or poor tissue penetration. In contrast, a nanobody (Nb) is a single-domain antibody of the variable domain of Camelid heavy-chain antibody, and it presents such unique properties as strong stability and specificity, high binding affinity, and good tissue penetration based on its small size (~15 kDa), making it an attractive therapeutic tool to prevent and treat human diseases. Our previous studies have established the technological platform for designing and developing novel and effective Nbs against Middle East respiratory syndrome coronavirus (MERS-CoV). Compared to conventional mAbs, these anti-MERS-CoV Nbs showed significantly enhanced stability and half-life. We have also demonstrated the ability of ZIKV E protein-based vaccines and therapeutics to protect against ZIKV infection in our established animal models. In the proposed study, we hypothesize that ZIKV E-specific Nbs, after appropriate engineering, will present extended half-life, excellent stability, good tissue penetration without toxicity, and improved efficacy against ZIKV infection. The specific aims are to (1) engineer ZIKV E-specific Nbs for generation of humanized Nbs with extended half-life and enhanced efficacy, (2) characterize these Nbs for stability and broad tissue permeability without toxicity, and (3) evaluate in vitro cross-neutralizing activity and in vivo efficacy of ZIKV E- specific Nbs against ZIKV infection. Within the tenure of this grant, the overall goal is to rapidly generate a novel and effective anti-ZIKV Nb with extended half-life, strong stability, excellent tissue penetration, and increased efficacy to prevent ZIKV infection. Even though ZIKV causes severe fetal damage and demise, and persists in neuronal and reproductive tissues for up to months with severe damage, the properties and characteristics of the final and fully developed Nb is expected to contribute to its efficacy in completely preventing the vertical transmission of ZIKV and clear it from infected tissues, leading to a significant reduction of ZIKV-associated microcephaly, brain abnormalities, other malformations and tissue damage. !